Basics of Chromatography

Introductory Principles
Chromatography is a combination of two
words;
* Chromo Meaning color
* Graphy Representation of something
on paper

History of Chromatography

Chromatography, literally "color writing", was first

employed
by
Russian
scientistMikhail
Tswettin
1903/1906. He continued to work with chromatography
in the first decade of the 20th century, primarily for the
separation
of
plantpigments
such
aschlorophyll,carotenes and xanthophylls. Since these
components have different colors (green, orange, and
yellow,respectively) they gave the technique its name.

 It is a physical separation method in which

the components of a mixture are separated
by differences in their distribution between
two phases, one of which is stationary
(stationary phase) while the other (mobile
phase) moves through it in a definite
direction. The substances must interact with
the stationary phase to be retained and
separated by it.
The stationary phase may be a solid, or a
liquid supported on a solid or gel, the mobile
phase may be either a gas or a liquid.

 Chromatograph: Instrument employed for a

chromatography
Eluent: Fluid entering a column
Eluate: Fluid exiting the column
Elution: The process of passing the mobile
phase through the column
Flow rate: How much mobile phase passed /
minute (ml/min)
Linear velocity: Distance passed by mobile
phase per 1 min in the column (cm/min)
Analyte: Itis the substance to be separated
during chromatography.

 Mobile Phase: Gas or liquid that carries the

mixture of components through the stationary
phase.
Stationary Phase (Immobilized phase): The part of
the apparatus that holds the components as
they move through it, separating them.
Retention time: Time taken for particular
analytes to pass through the system (from the
column inlet to the detector) under set
conditions.
Retardation factor: Fraction of an analyte in
the mobile phase of a chromatographic
system.

 Mixture of various components enters into

system at various rate of speed.
Mixture moves over the absorptive
materials & provides separation.
Repeated absorption takes place over
stationary bed which determines the rate.
Smaller the affinity of molecule to
stationary phase shorter time spent in a
column.

 Separates complex mixtures with precision.

Similar components can be separated
easily . Ex : proteins that vary by a single
amino acid
Purify any soluble substances if proper
conditions are employed.
Separates very delicate products.
Used in chemical or bio-processing field,
manufacturing plants.

Chromatography is used by scientists to:

Analyze to examine a mixture, its

components, and their relations to
one another
Identify to determine the identity of
a mixture or components based on
known components
Purify to separate components in
order to isolate one of interest for
further study

Based on the physical means by which

stationary (SP) and mobile phase (MP) comes
into contact
3 phases solid , liquid & gas.
MP can be either gas or liquid
SP can be either solid or liquid
Column chromatography: SP held in a narrow
tube through which MP is forced under
pressure.
Planar chromatography: SP supported on a flat
paper. MP moves through SP due to gravity.

Contd.
Based on MP used chromatography is classified
as
Gas Chromatography separates vaporized
samples with a carrier gas (mobile phase) and a
column composed of a liquid or of solid beads
(stationary phase) performed only on the
columns.
Liquid
Chromatography:
separates
liquid
samples with a liquid solvent (mobile phase) and
a column composed of solid beads (stationary
phase) performed either on surface or column.

Gas Chromatography

Introduction
Gas Chromatography: To separate stable
and volatile organic and inorganic compounds.
It involves a sample being vaporized and
injected onto the head of the chromatographic
column. The sample is transported through the
column by the flow of inert, gaseous mobile
phase. The column itself contains a liquid
stationary phase which is adsorbed onto the
surface of an inert solid.
Types : Gas Solid Chromatography (GSC)
Gas Liquid Chromatography (GLC)
or Gas Chromatography

TYPES OF GC
Gas-solid chromatography:
Here, the mobile phase is a gas while the
stationary phase is a solid.
Used for separation of low molecular gases,
e.g., air components, H2S, CS2 ,CO2 , rare
gases, CO and oxides of nitrogen

Gas-liquid chromatography:

The mobile phase is a gas while the

stationary phase is a liquid retained on the
surface as an inert solid by adsorption or
chemical bonding

Principle of Operation
The principle of separation in GC is partition
The mixture of component to be separated is
converted to vapor and mixed with gaseous mobile
phase.
The component which is more soluble in stationary
phase travel slower
and eluted later. The
component which is less soluble in stationary phase
travels faster and eluted out first.
Partition coefficient is the ratio of solubility of a
substance distributed between two immiscible
liquids at a constant temperature.
No two components has same partition coefficient
conditions. So the components are separated
according to their partition coefficient.

Instrumentation of GC
Carrier gas has pressure regulator and flow monitor

for constant flow of carrier gas- available in form of

compressed gas.
Sample injection port to introduce sample vapours
into gas stream - micro syringe
Columns with appropriate length of stationary phase
Thermal Compartment or thermostat to maintain
column at appropriate temperature
Detectors to detect the sample components as they
come out of the column.
Microprocessor or recorder to provide signal
proportional to amount of each component present
in analyte.

Carrier Gas
The gas is conducted from cylinder
through pressure and flow regulator to
sample injection port at a temperature T
Molecular sieve to remove water & other
impurities.
Flow rates monitored by rotameter.
Hydrogen,
helium,
argon,
nitrogen,
carbon-dioxide and air are used.
Hydrogen is advantageous but dangerous
Helium second best but expensive

Contd.
Nitrogen less expensive but
reduced sensitivity
Helium used due to the excellent
thermal conductivity, greater density
& allows more flow rate.
Hydrogen

better
thermal
conductivity and lower density hazardous

SAMPLE INJECTION SYSTEM

To determine the efficiency of the column.
Method of sample injection depends on
pressure in the column during introduction.
Liquid Samples : Microsringe can be used.
Sample is introduced into hot zone of the
column. So that liquid gets transferred into
gaseous phase.
Gas Samples : Tight syringe to deliver 0.1 10
ml of the sample. Rotary sample valve can be
used.
Solid Samples : Dissolved into volatile liquids
for introduction.

Chromatographic Column
Constructed using glass or metal tubing.
Types : Packed column and Capillary
column
Packed column:
glass or metal is used for packing
Length is 2 cm longer column is difficult to
pack
Forms can be U or helix or straight shaped
columns.
Inert gas support can be taken

Capillary Column
Open tubular columns made of fused silica.
Length 30 to 300 m and diameter of 1
mm
Thin walled column used for higher
efficiency due to reduced diameter that
reduces diffusion of molecules.

advantage is better separation of

components at lower temperature with
short time period.

Detectors
Located at the exit of separation column to
sense the presence of individual component as
they leave the column.
Short response time
Non-destructive of sample
Sensitivity, stability & linear response
Insensitive to flow rate of the samples
The detector then produces electrical signals
proportional to the concentration of the
components of solute.
The signals are amplified and recorded as
peaks at intervals on the chromatograph.

Thermal conductivity
Detector
TCD is based upon the fact that the heat
lost from a filament depends upon the
thermal conductivity of the stream of
surrounding gas as well as its specific
heat.

Contd.
When only carrier gas flows heat loss to
metal block is constant, filament T remains
constant.
When an analyte species flows past the
filament generally thermal conductivity
changes, thus resistance changes which is
sensed
by
Wheatstone
bridge
arrangement.
The
imbalance between control and
sample filament temperature is measured
and a signal is recorded.

Electron Capture Detector

Molecules of compounds, which posses

affinity for electrons, differ in their

electron absorbing capacities. This
difference is utilized in this detector for
identification of the compounds.
Working- A foil made up of a
radioactive metal like Ni63 (-emitter)
is placed inside a Teflon coated cell
which also contains a cathode and an
anode.

 In the absence of organic species, the

produced electrons migrate towards
positive electrode and produce a
certain constant standing current.
When a sample/eluent is present it
captures the electrons, elutes from
column, there is a drop in this constant
current.
The potential across two electrodes is
adjusted to collect all the ions and a
steady saturation current, is therefore,
recorded.

Flame Ionization Detector

This employs hydrogen flame that is maintained in
a small cylindrical jet made up of platinum or
quartz.
Effluent from the column with helium or nitrogen
as carrier gas are fed into the hydrogen flame,
gets ignited and undergoes pyrolysis to produce
ions.
For detection of these ions, two electrodes are
used that provide a potential difference.
The ions produced are repelled by the positive
electrode which hit the collector plate. The current
produced in doing so is amplified and fed to an
appropriate recorder.

Flame Ionization Detector

HPLC
HPLC stands for High-performance liquid
chromatography(sometimes referred to as
High-pressure liquid chromatography).
HPLC is a physical separation technique in
which a sample dissolved in a liquid is injected
into a column packed with small particles and it
is separated into its constituent components
HPLC is probably the most important and widely
used analytical technique for quantitative
analysis of organics and biomolecules
Most useful for pharmaceuticals, biomolecules,
and labile organics

Contd.
HPLC is a separation technique that involves
Injection of a small volume of liquid sample into a
tube packed with tiny particles (3 to 5 (m) in
diameter called the stationary phase)
Individual components of the sample are moved
down the packed tube (column) with a liquid
(mobile phase).
It is forced through the column by high pressure
delivered by a pump.
These components are separated from one
another by the column packing that involves
various chemical and/or physical interactions
between their molecules and the packing particles.

HPLC System

Contd
These separated components are detected
at the exit of this tube (column) by a flowthrough device (detector) that measures
their amount. The output from the detector
is called a liquid chromatogram
In principle, LC and HPLC work the same
way except the speed , efficiency,
sensitivity and ease of operation of HPLC is
vastly superior

HPLC Instrumentation
Overview

Principle Pattern

An Example
Solvent Reservoirs
Controller
Solvent Cabinet

Vacuum Degasser
Binary Pump

Autosampler
Thermostatted
Column Compartment
Detector

37

HPTLC
HPTLC is a sophisticated form of TLC.
Fastest of all chromatographic techniques.
Any combinations of stationary and mobile
phases can be used.
Analytical HPTLC is used for micro preparative
analysis (ie., separation of milligram scale for
analysis of fraction )
Gives more sharper and compact bands with
minimum distance of migration.
Used for both qualitative and quantitative
analysis.

HPLC Vs. HPTLC

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